This proposal describes our continuing efforts to determine the role played by pteridine and folate coenzymes or substrates in enzymatic systems. Structural factors commonly present in such naturally occurring pteridines include 2-amino and 4-hydroxy substituents, a reduced pyrazine ring and an alkyl substituent at the 5-position. During the course of a pteridine-mediated biological reaction, oxidation often occurs and in some cases there is also a loss of the 5-substituent. To investigate the biochemistry of these compounds the program has been divided into three major sections: (a) The efficient, quantitative isolation of pteridine requiring enzymes, such as the dihydrofolate and dihydropteridine reductases, and thymidylate synthetase, in bulk amounts for structural characterization purposes, using existing and new affinity chromatograhic procedures; (b) The use of fluorescent, photoactive, and mercurated substrate or cofactor analogs (e.g., fluorescein derivatives of Methotrexate (MTX) and 5-fluoro-2'-deoxyuridylate (FdUMP), or 5'azidophenol-FdUMP) of these enzymes, as structural probes; and (c) A study of the chemistry of the reduced and substituted pteridines using H and 13C nuclear magnetic resonance, and rapid reaction techniques (e.g., stop-flow spectrophotometry), with particular emphasis on the structural changes occurring when oxygen is incorporated, and on factors which induce lability into the 5-substituents. In addition the use of Methotrexate bound to a high MW polymer such as BSA is being investigated as a chemotherapeutic agent using the L1210 ascites tumor and Lewis lung carcinoma carried by BDF1 mice as typical animal models. BIBLIOGRAPHIC REFERENCES: Synthesis and Properties of 5-Methyldihydrofolate. T.L. Deits, A. Russell, K. Fujii and J.M. Whiteley. Chemistry and Biology of Pteridines (Wolfgang Pfleiderer, ed.) Walter de Gruyter, Berlin-New York (1976), p. 525. Direct Conversion of 4-Hydroxypteridines to their 4-Amino Analogs. G.R. Gapski and J.M. Whiteley. Chemistry and Biology of Pteridines (Wolfgang Pfleiderer, ed.) Walter de Gruyter, Berlin-New York (1976), p. 627.